Sam's Notebook

FastQ Trimming - Geoduck RNAseq Data Using fastp on Mox

by Sam White 6 min read

Per this GitHub Issue, Steven asked me to identify long non-coding RNA (lncRNA) in geoduck. The first step is to aggregate all of our Panopea generosa (Pacific geoduck) RNAseq data and get it all trimmed. After that, align it to the genome, followed by Ballgown expression analysis, and then followed by a variety of selection criteria to parse out lncRNAs.

Trimming was performed using fastp on Mox, along with FastQC and MultiQC. A list of the input files used can be found in the RESULTS section (see the linked MD5 files).

SBATCH script:

#!/bin/bash
## Job Name
#SBATCH --job-name=20220909-pgen-fastqc-fastp-mutliqc-rnaseq
## Allocation Definition
#SBATCH --account=coenv
#SBATCH --partition=coenv
## Resources
## Nodes
#SBATCH --nodes=1
## Walltime (days-hours:minutes:seconds format)
#SBATCH --time=4-00:00:00
## Memory per node
#SBATCH --mem=200G
##turn on e-mail notification
#SBATCH --mail-type=ALL
#SBATCH --mail-user=samwhite@uw.edu
## Specify the working directory for this job
#SBATCH --chdir=/gscratch/scrubbed/samwhite/outputs/20220909-pgen-fastqc-fastp-mutliqc-rnaseq

### FastQC and fastp trimming of P.generosa RNAseq data.

### fastp input filenames to be in format: *.fastq.gz



###################################################################################
# These variables need to be set by user

# Set FastQ filename patterns
fastq_pattern='*.fastq.gz'
R1_fastq_pattern='*R1*.fastq.gz'
R2_fastq_pattern='*R2*.fastq.gz'

# Set number of CPUs to use
threads=40

# Input/output files
trimmed_checksums=trimmed_fastq_checksums.md5
reads_dir=/gscratch/scrubbed/samwhite/data/P_generosa/RNAseq/
fastq_checksums=input_fastq_checksums.md5

# FastQC output directory
output_dir=$(pwd)

# Paths to programs
fastp=/gscratch/srlab/programs/fastp-0.20.0/fastp
fastqc=/gscratch/srlab/programs/fastqc_v0.11.9/fastqc
multiqc=/gscratch/srlab/programs/anaconda3/bin/multiqc

## Inititalize arrays
fastq_array_R1=()
fastq_array_R2=()
R1_names_array=()
R2_names_array=()
raw_fastqs_array=()


# Programs associative array
declare -A programs_array
programs_array=(
[fastqc]="${fastqc}"
[fastp]="${fastp}" \
[multiqc]="${multiqc}"
)


###################################################################################

# Exit script if any command fails
set -e

# Load Python Mox module for Python module availability
module load intel-python3_2017

# Capture date
timestamp=$(date +%Y%m%d)

# Sync raw FastQ files to working directory
echo ""
echo "Transferring files via rsync..."
rsync --archive --verbose \
"${reads_dir}"${fastq_pattern} .
echo ""
echo "File transfer complete."
echo ""

### Run FastQC ###

### NOTE: Do NOT quote raw_fastqc_list
# Create array of trimmed FastQs
raw_fastqs_array=(${fastq_pattern})

# Pass array contents to new variable as space-delimited list
raw_fastqc_list=$(echo "${raw_fastqs_array[*]}")

echo "Beginning FastQC on raw reads..."
echo ""

# Run FastQC
${programs_array[fastqc]} \
--threads ${threads} \
--outdir ${output_dir} \
${raw_fastqc_list}

echo "FastQC on raw reads complete!"
echo ""

### END FASTQC ###

# Create arrays of fastq R1 files and sample names
# Do NOT quote R1_fastq_pattern variable
for fastq in ${R1_fastq_pattern}
do
  fastq_array_R1+=("${fastq}")

  # Use parameter substitution to remove all text up to and including last "." from
  # right side of string.
  R1_names_array+=("${fastq%%.*}")
done

# Create array of fastq R2 files
# Do NOT quote R2_fastq_pattern variable
for fastq in ${R2_fastq_pattern}
do
  fastq_array_R2+=("${fastq}")

  # Use parameter substitution to remove all text up to and including last "." from
  # right side of string.
  R2_names_array+=("${fastq%%.*}")
done


# Create MD5 checksums for raw FastQs
for fastq in ${fastq_pattern}
do
  echo "Generating checksum for ${fastq}"
  md5sum "${fastq}" | tee --append ${fastq_checksums}
  echo ""
done


### RUN FASTP ###

# Run fastp on files
# Adds JSON report output for downstream usage by MultiQ
echo "Beginning fastp trimming."
echo ""

for index in "${!fastq_array_R1[@]}"
do
  R1_sample_name="${R1_names_array[index]}"
  R2_sample_name="${R2_names_array[index]}"
  ${fastp} \
  --in1 ${fastq_array_R1[index]} \
  --in2 ${fastq_array_R2[index]} \
  --detect_adapter_for_pe \
  --thread ${threads} \
  --html "${R1_sample_name}".fastp-trim."${timestamp}".report.html \
  --json "${R1_sample_name}".fastp-trim."${timestamp}".report.json \
  --out1 "${R1_sample_name}".fastp-trim."${timestamp}".fq.gz \
  --out2 "${R2_sample_name}".fastp-trim."${timestamp}".fq.gz

  # Generate md5 checksums for newly trimmed files
  {
      md5sum "${R1_sample_name}".fastp-trim."${timestamp}".fq.gz
      md5sum "${R2_sample_name}".fastp-trim."${timestamp}".fq.gz
  } >> "${trimmed_checksums}"
  
  # Remove original FastQ files
  echo ""
  echo " Removing ${fastq_array_R1[index]} and ${fastq_array_R2[index]}."
  rm "${fastq_array_R1[index]}" "${fastq_array_R2[index]}"
done

echo ""
echo "fastp trimming complete."
echo ""

### END FASTP ###


### RUN FASTQC ###

### NOTE: Do NOT quote ${trimmed_fastqc_list}

# Create array of trimmed FastQs
trimmed_fastq_array=(*fastp-trim*.fq.gz)

# Pass array contents to new variable as space-delimited list
trimmed_fastqc_list=$(echo "${trimmed_fastq_array[*]}")

# Run FastQC
echo "Beginning FastQC on trimmed reads..."
echo ""
${programs_array[fastqc]} \
--threads ${threads} \
--outdir ${output_dir} \
${trimmed_fastqc_list}

echo ""
echo "FastQC on trimmed reads complete!"
echo ""

### END FASTQC ###

### RUN MULTIQC ###
echo "Beginning MultiQC..."
echo ""
${multiqc} .
echo ""
echo "MultiQC complete."
echo ""

### END MULTIQC ###

####################################################################

# Capture program options
if [[ "${#programs_array[@]}" -gt 0 ]]; then
  echo "Logging program options..."
  for program in "${!programs_array[@]}"
  do
    {
    echo "Program options for ${program}: "
    echo ""
    # Handle samtools help menus
    if [[ "${program}" == "samtools_index" ]] \
    || [[ "${program}" == "samtools_sort" ]] \
    || [[ "${program}" == "samtools_view" ]]
    then
      ${programs_array[$program]}

    # Handle DIAMOND BLAST menu
    elif [[ "${program}" == "diamond" ]]; then
      ${programs_array[$program]} help

    # Handle NCBI BLASTx menu
    elif [[ "${program}" == "blastx" ]]; then
      ${programs_array[$program]} -help
    fi
    ${programs_array[$program]} -h
    echo ""
    echo ""
    echo "----------------------------------------------"
    echo ""
    echo ""
  } &>> program_options.log || true

    # If MultiQC is in programs_array, copy the config file to this directory.
    if [[ "${program}" == "multiqc" ]]; then
      cp --preserve ~/.multiqc_config.yaml multiqc_config.yaml
    fi
  done
fi


# Document programs in PATH (primarily for program version ID)
{
date
echo ""
echo "System PATH for $SLURM_JOB_ID"
echo ""
printf "%0.s-" {1..10}
echo "${PATH}" | tr : \\n
} >> system_path.log

RESULTS

Runtime was a bit over 3hrs.

NOTE: Job indicates it failed. Although technically true, the failure was specifically related to MultiQC trying to process a FastQC file that was run on an empty post-trimming FastQ file and accorred at the very end of the script. So, everything that “needed” to run actually did run; just don’t have a MultiQC summary for the FastQC analyses.

There was a single set of paired FastQs which had no sequence data after trimming:

Trueseq-stranded-mRNA-libraries-GeoRNA8-H1-NR021_S5_L001_R[12]_001.fastp-trim.20220908.fq.gz

Screencap of Mox job emails showing runtime of 3hrs 19mins and 5secs. Also shows job failed, but failure was simply due to an empty post-trimming FastQ file causing FastQC and, in turn MultiQC, to freak out.

Output folder:

All trimmed FastQs can be retrieved with this pattern:

*fastp-trim.20220908.fq.gz